Browsing your Data
Once you have the DEBrowser running, a page will load asking to choose a CSV file or to load the demo data. In order to run DESeq2, we are going to need gene quantifications for those genes contained in a comma-seperated values (CSV) or tab-seperated values (TSV) format. The file values must contain the gene, transcript, and the samples count values you wish to enter into DEBrowser.
IE:
# CSV:
gene,transcript,exper_rep1,exper_rep2,control_rep1,control_rep2
DQ714826,uc007tfl.1,0.00,0.00,0.00,0.00
DQ551521,uc008bml.1,0.00,0.00,0.00,0.00
AK028549,uc011wpi.1,2.00,1.29,0.00,0.00
# TSV:
gene transcript exper_rep1 exper_rep2 control_rep1 control_rep2
DQ714826 uc007tfl.1 0.00 0.00 0.00 0.00
DQ551521 uc008bml.1 0.00 0.00 0.00 0.00
AK028549 uc011wpi.1 2.00 1.29 0.00 0.00
You can also view/use the demo data by clicking the ‘Load Demo!’ text as an example. For the case study demo data, feel free to download our case study demo file at http://galaxyweb.umassmed.edu/pub/DC/advanced_demo.tsv After obtaining and loading in the gene quantifications file, you are then able to view QC information of your quantifications or to continue on to running DESeq2.
Figure 1: The initial options selection.
Upon selection of QC information, you will be shown an all-to-all plot of your samples. This sample-by-sample comparison will help you visualize possible descrepencies between replicate samples, in case you may want to omit them for further analysis. To the left of this plot are various plot-shaping options you can alter to more easily view the all-to-all plot.
Additionally, two addition QC plots are available for you to use: Heatmap and PCA plots. The heatmap will display genes for each sample within your dataset in the form of a heatmap and PCA will display Principal component analysis of your dataset. You can select these various plot options by selecting the type of plot you wish to view on the left panel.
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Figure 2: Display of the all-to-all plot in the initial QC plots page.
You can also view the genes within your quantification file in various ways. The ‘All Detected’ tab will list all of the genes present within your file. The ‘Gene Set’ tab displays all of the genes within a specific search. You can enter genes into the search box by selecting the ‘geneset’ dropdown option on the left. The Last tab ‘Most Varied’ will display your top N varied genes. You can alter the value of N by selecting ‘most-varied’ from the dropdown menu on the left.
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Figure 3: Display of the heatmap in the initial QC plots page.
Figure 4: Display of the PCA plot in the initial QC plots page.
Figure 5: Display of most varied genes.
Upon selecting to run DESeq, you are then able to select which samples will be selected for your first condition and second condition to use for differential expression analysis. By clicking the ‘Add New Comparison’ button, you can add as many different comparisons as you want. Sample names are created based on the column headers within your data file. Once you’ve selected your comparisons, you are then ready to run DESeq2 to calculate differential expression by clicking on the ‘Submit!’ button.
Figure 6: Menus after loading in a sample.
Analyzing the Results
After clicking on the ‘Submit!’ button, DESeq2 will analyze your comparisons and store the results into seperate data tables. Shiny will then allow you to access this data, with multiple interactive features, at the click of a button. It is important to note that the resulting data produced from DESeq is normalized. Upon finishing the DESeq analysis, a tab-based menu will appear with multiple options.
Figure 7: List of the tabbed menus in DEBrowser.
The first tab, the ‘Main Plots’ section, is where you will be able to view the interactive results plots. On the left hand side of the screen will be the options you have to alter the padj and fold change cutoff values, what specific data set to use such as up or down regulated genes, what comparison dataset you would like to use to plot, and what type of plot you would like to view your results in. Plot choices include:
Main plot
Volcano plot
MA plot
Figure 8: Main scatter plot and the zoomed in main scatterplot.
Figure 9: Main volcano Plot and the zoomed in main volcano plot.
Figure 10: Main MA plot and the zoomed in main MA plot.
Once you have selected your values, you can hit the ‘Submit!’ button to create your interactive plots!
The top left plot is whichever plot you have selected to use to analyze your results. Up-regulated genes are displayed in green while down-regulated genes are displayed in red. Hovering over a gene on this plot will display the bottom two plots: the genes normalized variation and colored by condition in the left graph, and the normalized variation between conditions within the right graph. Hovering over a gene will also display information about that gene in regards to both conditions you have selected. By clicking and dragging your mouse to create a selection over the main graph, you will create the top right plot, or the zoomed in version of your selection. If you are going to change any of the parameters on the left, please make sure to re-click the ‘Submit!’ button to update the graphs. It’s also worth noting that the plots are resizable as well as downloable.
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Figure 11: The main plots page within DEBrowser.
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Figure 12: Display of the most varied genes as a scatter plot.
Figure 13: Display of the geneset list as a scatter plot.
Selecting the ‘QC Plots’ tab will take you to the quality control plots section. These QC plots are very similar to the QC plots shown before running DESeq, however the dataset being used here depends on the one you select on the left menu. In addition to the all-to-all plot shown within the previous QC analysis, users can also view a heatmap and PCA plot of their analyzed data by selecting the proper plot on the left menu.
Figure 14: Display of the heatmap within DEBrowser.
The heatmap is a great way to analyze replicate results of genes all in one simple plot. Users have the option to change the clustering method used as well as the distance method used to display their heatmap. In addition, you can also change the size of the heatmap produced and adjust the p-adjust and fold change cut off for this plot as well. Once all of the parameters have been set, click the ‘Submit!’ button at the bottom of the left menu to generate your heatmap.
You can also select to view an interactive version of the heatmap by clicking on the ‘Interactive’ button on the left panel under the height and width options. Selecting this feature changes the heatmap into an interactive version with two colors, allowing you to select specific genes to be compared within the GO term plots. This will allow you to compare interesting clusters found within the the heatmap within our GO Term analysis section which will be discussed later within the materials.
Figure 15: View of the interactive Heatmap
Figure 16: Display of the PCA plot within DEBrowser.
Prinicipal Component Analysis (PCA) is another excellent method of checking replicates. PCA calculates the variance between all of the samples genes within your current comparison set and creates a two-dimensional graph to represent the proportion of variance explained in different components. Within the PCA plot section you can select the p-adjust value, fold change cut off value, which comparison set to use, which dataset to use, the height and width of the corresponding plots, as well as which prinicipal components to analyze by changing the appropriate values on the left menu.
The next tab, ‘GO Term’, takes you to the ontology comparison portion of DEBrowser. From here you can select the standard dataset options such as p-adjust value, fold change cut off value, which comparison set to use, and which dataset to use on the left menu. In addition to these parameters, you also can choose from the 4 different ontology plot options: ‘enrichGO’, ‘enrichKEGG’, ‘Disease’, and ‘compareCluster’. Selecting one of these plot options queries their specific databases with your current DESeq results. By selecting the ‘selection’ dataset on the left panel after selecting specific genes from the interactive heatmap, you will be able to compare your specific gene selection within the various GO Term databases.
Figure 17: Display of the GO Plot section within DEBrowser.
Figure 18: Display of the GO dotplot section within DEBrowser.
Figure 19: Display of the DO plot section within DEBrowser.
Figure 20: Display of the DO dotplot section within DEBrowser.
Figure 21: Display of the KEGG plot section within DEBrowser.
The next six tabs contain various result information in table formats. The ‘All Detected’ tab contains the list of all the genes within the TSV/CSV provided with the corresponding DESeq analyses. Up-regulated values are shown in green while down-regulated values are displayed in red. To view any particular tab’s custom options, the dataset type in the left panel must match that of the tab you are viewing.
The ‘Up’ tab contains a list of all the up-regulated genes based on the options selected on the left panel. The ‘Down’ tab contains a list of all the down-regulated genes based on the options selected. The ‘Selected’ tab contains the list of genes selected from the main plots section. By clicking and dragging your mouse on the main plot within the ‘Main Plots’ tab, you will then be able to see that selection in list form within the ‘Selected’ tab. The ‘Gene Set’ tab allows you to filter out gene data based on genes selected via a text box. To create a gene set, select the ‘geneset’ option on the left panel under the ‘Choose a dataset’ option. Once selected, a text box will appear where you can input various genes of interest. You can also use regular expressions in order to search for specific gene sets. The ‘Most Varied’ tab, much like the original QC ‘Most Varied’ tab, allows you to view the list of most varied genes based on user input parameters on the left panel. The Last tab, ‘Comparisons’, allows you to view the differences between your different condition comparisons. Comparisons between datasets are shown if at least one of the conditional comparisons has passed the padj value or fold change cut off.
Make sure that when you are comparing/viewing tables within the specified tables tabs, that you select the correct dataset you would like to used on the left panel. It is also important to note that comparisons with only one sample cannot create statistically significant p-adjust values. The more replicates you have within a condition, the greater the statistical significance of your comparisons.
Figure 22: Display of the up+down-regulated genes table.
Figure 23: Display of the down-regulated genes table.
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Figure 24: Display of the geneset input box.
Figure 25: Display of the gene set search of the term ‘^al’.
Figure 26: Condition comparisons table within DEBrowser.
Lastly, the tables have a bunch of features that allow you to view your DESeq results more conviently. By clicking on a column header, you can sort the data within the table based either alphabetical or numeric sorting. You can also enter a term within the search box to search for a specific gene within the table.
With that, you’ve now successfully navigated the DEBrowser and are ready to start inserting your own data files and browsing your own experiments. Enjoy the DEBrowser!